DESCRIPTION: This project will investigate the molecular pathogenesis of Angelman syndrome (AS) by analyzing a strong candidate gene, E6-AP. AS is a mental retardation syndrome well known for its distinctive inheritance, which indicates an imprinting effect. The majority of AS patients have maternal deletions of 15q11-13 (paternal deletions of the same region are associated with Prader-Willi syndrome). A small proportion of AS patients have paternal uniparental disomy of chromosome 15 or a paternal pattern of methylation on both chromosomes. Thus, the disease appears to be due to loss of expression of a maternally imprinted gene. About 25 percent of AS patients do not have detectable deletion, uniparental disomy, or methylation abnormality. Families of patients in this category display the AS phenotype with maternal and not paternal transmission. Over the past several years, the AS locus at 15q has been narrowed by mapping deletions and recombinations to a narrow segment between D15S122 and D15S10. E6-AP is a ubiquitin-protein ligase originally identified as mediating the interaction of the human papilloma virus protein E6 with p53. A previous study mapped E6-AP to 15q near the AS critical region, but expression analysis in lymphoblasts and fibroblasts showed no evidence of parental imprinting and no abnormality of expression in AS or Prader-Willi syndrome. However, when the applicants did exon-trapping with a P1 clone covering the critical region, E6-AP exons were recovered. In a study that was just recently published in Nature Genetics, they showed the E6-AP gene to span a translocation breakpoint in one AS family and to have frameshifting mutations in two others. Another group has also reported E6-AP mutations in AS families just recently. In the proposed project, E6-AP will be further characterized as a candidate gene for AS: (1) by seeking mutations in other non-deletion AS patients through use of SSCP and RNase cleavage analysis, (2) by determining the effects of the mutations on E6-AP ubiquitination using an in vitro assay, (3) by looking for genotype-phenotype correlation, (4) by isolating and characterizing the 5' end of the E6-AP transcript, (5) by identifying polymorphisms in the transcript for use in imprinting analysis of human tissue samples, (6) by looking for differential methylation in AS and Prader-Willi patient DNA, and (7) by seeking interacting proteins through use of the yeast two-hybrid system.